Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disease characterized by varying degrees of weakness of the skeletal muscles of the body. The most common cause of MG is the presence of auto-antibodies against the nicotinic acetylcholine receptor (AChR) of the muscle.
AChR is a pentameric, transmembrane protein composed of five subunits in a stoichiometry of α2, β, γ, and δ. In adults, the receptor γ subunit is replaced by the ε-subunit. AChR are located at high density in the postsynaptic membrane of the neuromuscular junction (NMJ). After binding of acetylcholine (ACh), released by the nerve terminal, AChR facilitate the depolarisation of the postsynaptic membrane, leading to the contraction of muscle fibers. Anti-AChR antibodies induce a loss of AChR, leading to an impaired neuromuscular transmission. This results in fluctuating skeletal muscle weakness that worsens with use, and improves with rest. If the concentration of AChR is very low, the effects can also be measured by electrophysiology, showing a decrementing response of the compound muscle action potential (CMAP) after repetitive nerve stimulation.
Anti-AChR antibodies of all IgG subclasses have been found in MG patients, but the only isotype that is always present is IgG1 (Rodgaard, A. et al., Clin Exp Immunol 67, 82 (1987)). Pathogenic mechanisms include: damage of the NMJ by focal lysis of the postsynaptic membrane due to complement activation, cross-linking of AChRs, which leads to increased internalization and degradation of the receptors (antigenic modulation) (Heinemann, S. et al., Proc Natl Acad Sci USA 74, 3090 (1977)) and Kao, I. et al., Science 196, 527 (1977), inhibition of or ion channel function (Lang, B. et al., J Neuroimmunol 19, 141 (1988)) and blocking of the ACh binding site (Almon, R R. et al., Science 186, 55 (1974)). The extracellular domain of the α subunits contains the main immunogenic region (MIR), to which a major part of the pathogenic MG antibodies is directed (Tzartos, S J et al., Proc Natl Acad Sci USA 77, 755 (1980), Tzartos, S J et al., Proc Natl Acad Sci USA 79, 188 (1982) and Tzartos, S J. et al., Immunol Rev 163, 89 (1998). Anti-MIR antibodies are also very effective in antigenic modulation.
The effects of auto-antibodies directed to the proteins of the NMJ can be studied in an animal model of MG, termed experimental autoimmune myasthenia gravis (EAMG). The original experimental model, which led to the discovery of the cause of MG, is the immunization of rabbits with the AChR from the electric organ of Electrophorus electricus which induced a chronic EAMG (Patrick, J. et al., Science 180, 871 (1973). Subsequently, the similarity to human MG was demonstrated by immunising rhesus monkeys (Macaca mulatta) with repeated doses of Torpedo californica AChR (Tarrab-Hazdai, R. et al., Nature 256, 128 (1975)). Electromyography showed a decreased action potential after repetitive nerve stimulation, confirming impaired neuromuscular transmission. In a rodent chronic EAMG model, a sub-population of antibodies against the Torpedo or Electrophorus AChR that cross-react with the AChR of the immunised animal were identified as the cause of the damage to the NMJ, leading to a long-lasting disease (Lindstrom, J M. et al., Ann N Y Acad Sci 274, 254 (1976). Sera from MG patients, AChR-immunised animals and monoclonal anti-AChR antibodies have also been shown to induce MG symptoms when injected in naïve animals (passive transfer EAMG) (Toyka, K V et al., Science 190, 397 (1975), Toyka, K V et al., N Engl J Med 296, 125 (1977) and Lindstrom, J M et al., J Exp Med 144, 739 (1976)). Passive transfer causes a reversible muscular weakness that lasts for several days.
Several specific immunotherapy approaches have been tested in EAMG models, including anti-idiotypic Abs, mAb-competing peptide, tolerance induction, and elimination of AChR-specific T cells by genetically engineered antigen-presenting cells (Souroujon, M C et al., Neurology 36, 622 (1986), Verschuuren, J J et al., J Immunol 146, 941 (1991), Luo, G X et al., J Immunol Methods 251, 177 (2001), Wang, Z Y et al., J Neuroimmunol 44, 209 (1993), Im, S H et al., Faseb J 15, 2140 (2001), Wu, J M et al., Cell Immunol 208, 137 (2001)). None of these approaches have lead to an efficient MG therapy so far.
An alternative approach is the direct blocking of the auto-antibody binding sites of the AChR. This approach is feasible since a large fraction of the circulating autoantibodies in MG patients is directed against the MIR. It has been shown that a patient derived anti-AChR Fab-637 is capable of blocking the binding of serum derived polyclonal anti-AChR antibody derived from various unrelated MG patients to human AChR in vitro (Graus, Y F et al., J Immunol 158, 1919 (1997)). As anti-MIR Fab fragments do not have intrinsic properties to induce loss of functional AChR, since they do not activate complement, induce antigenic modulation or functionally inhibit the AChR, anti-MIR Fab fragments have been successfully used to prevent passive transfer of EAMG in rats and mice (Loutrari, H. et al., Eur J Immunol 22, 2449 (1992), Toyka, K V. et al., J Neurol Neurosurg Psychiatry 43, 836 (1980) and Papanastasiou, D K et al., J Neuroimmunol 104, 124 (2000). Human anti-human AChR Fab fragments have been cloned and their ability to block MG serum Abs explored (Rey, E. et al., Clin Immunol 96, 269 (2000), Farrar, J. et al., Int Immunol 9, 1311 (1997), Protopapadakis, E. et al., Eur J Immunol 35, 1960 (2005) and Stassen, M H et al., J Neuroimmunol 135, 56 (2003). However, no further in vivo proof of concept was obtained and Fab molecules are unsuitable for treatment of patients, inter alia due to their short half-life.
In general, five methods of treatment are currently used in MG (Sieb, J P, Curr Opin Pharmacol 5, 303 (2005): 1) Enhancement of neuromuscular transmission using acetylcholinesterase (AChE) inhibitors, such as neostigmine and pyridostigmine, prolonging the action of acetyl choline, which helps improve neuromuscular transmission and increase muscle strength, 2) immunosuppression, using drugs such as prednisone, cyclosporine, and azathioprine, aiming to suppress the production of auto-antibodies, 3) thymectomy, 4) elimination of auto-antibodies by plasma exchange, and 5) modulation of the autoimmune response by intravenous immunoglobulins.
However, none of these methods of treatment is very efficacious or specific and thus, there is a need for improved methods for treating myasthenia gravis and other auto-immune diseases.